Solid-phase polymer synthesis on reusable substrates

Abstract

Substrates for solid-phase synthesis are reused by freeing synthesized polymers without removing the linkers that hold the polymers to the substrate. The linkers may be made of oligonucleotides or polypeptides. In an implementation, the polymers are released by cleavage of the linkers and then the truncated linkers are regenerated by adding back the portion that was removed. In an implementation, molecular bonds between the linkers and the polymers are cleaved releasing the polymers while leaving the linkers available for reuse without regeneration. In an implementation, single-stranded oligonucleotide linkers are hybridized to complementary strands that hold the polymers to the substrate with double-stranded oligonucleotide complexes. The double-stranded oligonucleotide complexes are denatured releasing the polymers while leaving the original linkers attached to the substrate. The polymers that are synthesized with these techniques may be the same or different type of molecules than the linkers.

1 . A method for solid-phase synthesis of polymers, the method comprising:
 generating polymer strands by adding monomers to free ends of linkers attached to a substrate;   contacting the linkers with a linker cleavage agent that cleaves the linkers at a recognition site thereby releasing the polymer strands from the substrate and generating truncated linkers; and   regenerating the linkers from the truncated linkers.   
     
     
         2 . The method of  claim 1 , wherein the linkers comprise single-stranded oligonucleotide linkers and the linker cleavage agent comprises a restriction endonuclease that cleaves the linkers within the recognition site. 
     
     
         3 . The method of  claim 2 , further comprising, prior to contacting the linkers with the linker cleavage agent, contacting the linkers with linker complement strands under conditions that cause the linker complement strands to hybridize with the linkers, wherein the recognition site is a double-stranded oligonucleotide sequence formed from hybridization of the linkers and the linker complement strands. 
     
     
         4 . The method of  claim 1 , wherein the linkers comprise single-stranded oligonucleotide linkers and regenerating the linkers comprises:
 contacting the truncated linkers with regeneration templates under conditions that cause the regeneration templates to hybridize with the truncated linkers; and   (i) extending the truncated linkers by addition of polymerase and nucleotides; or   (ii) contacting the regeneration templates with linker replacement strands and ligase.   
     
     
         5 . The method of  claim 1 , wherein the linkers comprise polypeptide linkers and the linker cleavage agent comprises a protease that cleaves the linkers at the recognition site. 
     
     
         6 . The method of  claim 1 , wherein the linkers comprise polypeptide linkers and the linker cleavage agent comprises a chemical cleavage agent. 
     
     
         7 . The method of  claim 1 , wherein the linkers comprise polypeptide linkers and regenerating the linkers comprises performing solid-phase synthesis of polypeptides to extend the truncated linkers. 
     
     
         8 . The method of  claim 1 , wherein the polymer strands comprise oligonucleotides and the method further comprises removing scars from the polymer strands by amplification with polymerase chain reaction (PCR). 
     
     
         9 . A method for solid-phase synthesis of polymers, the method comprising:
 a. generating polymer strands by adding monomers to free ends of linkers attached to a substrate;   b. contacting the linkers with a linker cleavage agent that cleaves at points of attachment between the linkers and the polymer strands; and   c. washing the substrate to remove the polymer strands without removing the linkers from the substrate.   
     
     
         10 . The method of  claim 9 , further comprising repeating steps a-c at least once. 
     
     
         11 . The method of  claim 9 , wherein the linkers comprise single-stranded oligonucleotide linkers and the method further comprises, prior to contacting the linkers with the linker cleavage agent, contacting the linkers with linker complement strands under conditions that cause the linker complement strands to hybridize with the linkers. 
     
     
         12 . The method of  claim 11 , wherein the linker cleavage agent comprises a restriction endonuclease having a recognition site within a double-stranded oligonucleotide sequence formed by hybridization of the linkers with the linker complement strands and a cleavage site at the point of attachment of the linkers to the polymer strands. 
     
     
         13 . The method of  claim 11 , wherein the linkers comprise deoxyribonucleic acid (DNA) strands having uracil bases at the free ends and the linker cleavage agent comprises Uracil DNA. Glycosylase (UDG). 
     
     
         14 . The method of  claim 9 , wherein the linkers comprise poly peptide linkers and the linker cleavage agent comprises a protease that cleaves at the points of attachment between the polypeptide linkers and the polymer strands. 
     
     
         15 . A method of solid-phase synthesis of oligonucleotides, the method comprising:
 contacting single-stranded oligonucleotide linkers attached to a substrate with adapter strands under conditions that cause the adapter strands to hybridize with the single-stranded oligonucleotide linkers;   generating oligonucleotide strands that are (i) covalently attached to the adapter strands or (ii) partially hybridized to the adapter strands; and   releasing the oligonucleotide strands from the substrate by denaturing the single-stranded oligonucleotide linkers and the adapter strands.   
     
     
         16 . The method of  claim 15 , wherein the single-stranded oligonucleotide linkers are oriented with 3′-ends attached to the substrate and free 5′-ends; and
 generating the oligonucleotide strands that are (i) covalently attached to the adapter strands comprises extending the adapter strands by non-templated oligonucleotide synthesis thereby creating the oligonucleotide strands. 
 
     
     
         17 . The method of  claim 15 , wherein the single-stranded oligonucleotide linkers are oriented with 5′-ends attached to the substrate and free 3′-ends, wherein the 3′-ends are modified to prevent extension, and generating the oligonucleotide strands (ii) partially hybridized to the adapter strands comprises:
 generating anchoring oligonucleotide strand fragments complementary to overhanging regions of the adapter strands; and 
 extending the anchoring oligonucleotide strand fragments by non-templated oligonucleotide synthesis thereby creating the oligonucleotide strands. 
 
     
     
         18 . The method of  claim 17 , wherein the generating the anchoring oligonucleotide stand fragments comprises:
 contacting the adapter strands with polymerase and nucleotides; or   contacting the adapter strands with pre-synthesized oligonucleotide strands under conditions that cause the pre-synthesized oligonucleotide strands to hybridize with the overhanging regions of the adapter strands.   
     
     
         19 . The method of  claim 15 , wherein the substrate comprises glass or silicon and functionalization comprising silane groups or agarose. 
     
     
         20 . The method of  claim 15 , further comprising, washing the substrate to remove the oligonucleotide strands without removing the single-stranded oligonucleotide linkers.